Bispecific antibodies: design, therapy, perspectives

Sedykh, Sergey E; Prinz, Victor V; Buneva, Valentina N; Nevinsky, Georgy A

Bispecific antibodies: design, therapy, perspectives the second heavy chain with mutations T366S, L368A, or Y407V ("hole": replacement by a smaller amino acid); formation of heterodimers is thermodynamically favorable. 101, 102 A variant of method using structural similarity of IgG and IgA CH3 domains ensures the formation of heterodimers of heavy chains. 103 Several methods of BsAb production use just one type of light chain. 56 However, the most widely used method of BsAb production today is expression of monospecific mAbs in two different cell lines, isolation and subsequent combination in vitro. 104, 105 The advantage of this approach is using well-characterized Abs, but the significant disadvantage is the high cost and difficulty of obtaining such BsAbs. 2 BsAbs containing a paratope recognizing two different antigens have been demonstrated for an anti-HER2 Ab binding VEGF 106 and anti-HER3 Ab binding EGFR. 107 CrossMAb technology, developed by Roche, made it possible to generate tetraspecific Abs binding EGFR, HER2, HER3, and VEGF. 78 DutaMab technology (Creative Biolabs and Roche) uses three complementarity-determining regions (CDRs) in each antigen-binding site to bind one and three other CDRs to bind the second antigen, thus forming two paratopes. This technology allows BsAbs to be produced using methods typical for monospecific Abs, but the drawback of this approach is its nonuniversality: it is not possible for each pair of antigens to match the paratopes within a single antigen-binding site. 2 BsAbs that do not contain constant regions have been described, with the most popular and demanded formats diabody and BiTE. Such BsAbs are expressed in one cell, where the fragments of heavy and light chains are connected by short peptide sequences. ScFvs are widely used for generation of such BsAbs. For diabody generation, sequences encoding two different scFvs are combined into one construct in which heavy chains are expressed in a single polypeptide and then joined with the corresponding light chains. The first described diabodies were bivalent BsAbs. 108 Heterodimeric constructs were later obtained by the knob-in-hole method 109 and by single-chain diabody generation. 110, 111 Diabody technology was used to construct the first BiTEs in eukaryotic cells. BiTEs are small scFv molecules tandem connected by flexible peptide linkers. BiTEs usually contain an antigenbinding site against CD3 and another against a high-affinity surface antigen of tumor cells. 44, 112, 113 A disadvantage of such molecules is their short life span in blood serum, which is associated with smallness and lack of Fc. The advantage of this BsAb format is extremely highly specific antitumor activity at concentrations up to 10 pg/mL in cell culture. 114 According to some data, one BiTE molecule can be involved several times in elimination of tumor cells by cytotoxic T lymphocytes. 115 Single-domain Ab fragments obtained from mouse and human libraries by a phage display are also used to construct BsAbs. 116, 117 Nanobodies derived from llamas and camels contain only heavy chains; for the production of BsAbs, nanobodies are readily connected by short peptide linkers. 118 The advantages of using single-domain Ab fragments are their smallness, easy penetration into cells, and access to antigens hidden for IgG. A significant disadvantage of such small structures is their low half-life in the blood, which requires frequent injection of the drug. 2 The most successful re